Neurons are extremely polarized cells, and this polarity is crucial for their function. Dendrites receive signals and axons send them. One of the most basic differences between axons and dendrites, that could be the foundation for their important functional differences, is polarity of the microtubule (MT) cytoskeleton. As MTs have intrinsic polarity that is read by motor proteins, MT polarity is likely to be extremely important for polarized neuronal trafficking. However, mechanisms that control neuronal MT polarity are poorly understood. We will use a simple Drosophila model system to study this problem. In all systems axonal MTs are oriented with plus ends distal to the cell body (plus-end-out). Dendrites are distinguished by the presence of minus-end-out MTs. In cultured mammalian neurons, dendritic MTs have mixed polarity. But in vivo in Drosophila, and perhaps in mammalian neurons, dendritic MTs have essentially uniform polarity that is opposite of axons (minus-end-out). In this proposal we will focus on two particularly understudied aspects of neuronal MT polarity: establishment of a uniform minus-end-out dendritic MT array, and the organization of MTs in branched regions of axons. As no mechanistic studies on uniform minus-end-out dendritic MTs had been performed, we began our studies with close observation of dendritic MTs in vivo. This allowed us to hypothesize that MT growth must be directed in dendrites to maintain uniform polarity. We have now confirmed this hypothesis and identified KIF3 as a key player in directed MT growth that is required for minus-end-out polarity. In this proposal we will build upon this novel idea of directed MT growth by identifying proteins that allow KIF3 to interact with growing MTs and by determining where in dendrites it acts. In addition to continuing to study maintenance of dendritic MT polarity, we will investigate how minus- end-out polarity is established by focusing on the minus ends. It is not known whether dendritic MT minus ends are focused at a known microtubule organizing center (MTOC), for example the Golgi complex. We will investigate the role of known MTOCs by removing them from dendrites and assaying MT organization. We will also identify the pathways that generate minus ends in dendrites: nucleation only, or severing existing microtubules. Identifying the pathway responsible for making minus ends is crucial for understanding how a minus-end-out MT array is generated and controlled. Having established assays to study neuronal microtubule polarity in vivo, we will extend our analysis to a region of the cell which we have not yet examined: the distal branched region of axons. Precise MT organization in distal axons could be extremely important for synaptic function. The proposed studies will provide major insight into mechanisms that control the tracks for long-range neuronal transport. By focusing on poorly studied dendrites and distal axons we will have maximum impact. PUBLIC HEALTH RELEVANCE: Microtubules are the tracks for long-range cellular transport, and they are particularly important for the function of elongated neuronal cells, which have specific arrangements of microtubules in axons and dendrites. We will identify molecular mechanisms required for organization of microtubules using Drosophila neurons as a simple, but extremely powerful, model system. Our results will form a foundation for understanding neurological diseases ranging from motor neuron disease to Williams syndrome, as they result from perturbations in microtubule organization or trafficking.